Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/46—Cellular immunotherapy
  • A61K39/461—Cellular immunotherapy characterised by the cell type used
  • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/46—Cellular immunotherapy
  • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
  • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/46—Cellular immunotherapy
  • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
  • A61K39/4643—Vertebrate antigens
  • A61K39/46433—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
  • A61K2239/26—Universal/off- the- shelf cellular immunotherapy; Allogenic cells or means to avoid rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
  • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
  • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

• the present invention relates to the fields of immunology and medicine. It relates more particularly to a new immunological approach allowing the treatment of many pathologies.
• the invention is based more particularly on a procedure "exchange of T lymphocytes" for therapeutic purposes, after depletion of the T lymphocytes of the host.
• the invention can be applied to the treatment of numerous pathologies, in which T lymphocytes are involved, such as autoimmune diseases, transplants, viral diseases, allergy, etc.
• T lymphocytes are essential cells for developing effective immune responses that help protect the body, especially against infectious agents or tumor cells.
• the response of T lymphocytes is most often beneficial, in certain situations it can on the contrary be a source of pathologies (autoimmune diseases, rejection of transplants, etc.). In these situations it is therefore particularly important to be able to control the response of T lymphocytes in order to treat immunopathology.
• Organ transplant suites offer a good example of the use of these different treatments.
• Organ transplants are always followed by treatments with so-called conventional immunosuppressants such as PEndoxan, corticosteroids, cyclosporine or FK506, intended to protect the graft from the host's immune response which leads to rejection. While these treatments have proven to be relatively effective, as evidenced by the progress made in organ transplants, they do not always succeed in preventing rejection of transplants.
• T lymphocytes polyclonal sera or monoclonal antibodies recognizing surface molecules expressed on the surface of T lymphocytes (anti-CD3, anti-CD4). These treatments act by destroying all T lymphocytes, whether those responsible for transplant rejection like all other T lymphocytes, and therefore also have a lack of specificity, inducing a significant state of immunosuppression in the subjects.
• GMO autologous bone marrow transplantation
• Allogenic GMO is now proposed for some of these pathologies such as severe autoimmune pathologies, with the aim of reconstituting the patient with different lymphocytes which should not participate in the immunopathological reactions.
• allogeneic bone marrow transplantation is marred by significant morbidity due to GVH, which limits its use.
• the T lymphocytes present in the hematopoietic graft and reinjected into an immunosuppressed patient due to the conditioning necessary for the graft will be responsible for an attack directed against the cells of the host also called graft reaction against the host (GVH).
• This reaction is sometimes difficult to control with conventional immunosuppressive treatments and is sometimes even fatal. It is not legitimate today to offer a patient with chronic joint disorders, but who may develop over several years or even several decades, a treatment with a mortality greater than 10%.
• the present invention represents a new therapeutic approach for the regulation of the immune response.
• the present invention relates more particularly to a new concept of modulation of the immune response of T lymphocytes, in particular of the immunopathological T lymphocyte response.
• the invention has numerous advantages over the approaches described in the prior art, in particular in terms of specificity, stability (duration) and comfort for the subjects.
• a first aspect of the invention relates more particularly to a method of modifying the immune system of a subject comprising the replacement of the T lymphocytes of this subject. More particularly, this method comprises the depletion of T lymphocytes (or subpopulations of T lymphocytes) of the subject (without depletion of other hematopoietic cells, including stem cells), followed by the administration of a composition comprising T lymphocytes modified (or T cell subpopulations), autologous, syngeneic or allogenic. This method is more particularly intended to control the immunopathological responses mediated by T lymphocytes.
• Another particular aspect of the invention also relates to a method for regulating the immunopathological responses induced by T lymphocytes, comprising the replacement of all or part of the T lymphocytes of a subject by a population of modified T lymphocytes.
• the invention also relates to a method of treatment of pathologies caused by T lymphocytes, comprising the replacement of all or part of the T lymphocytes of a subject by a composition comprising modified T lymphocytes.
• the invention also relates to the use of a population of T lymphocytes for the preparation of a composition intended to be administered to a subject having undergone depletion of all or part of his T lymphocytes.
• the invention also relates to the preparation of 'a composition of T lymphocytes whose repertoire has been modified (enriched in or, on the contrary, deviated from certain antigenic specificities).
• the present invention therefore relates to a new medical approach, based on the replacement of a subject's T lymphocytes, and often referred to in the following as the expression "T lymphocyte exchange”.
• the present invention is based in part on a new concept of T-cell homeostasis.
• the invention is based on the notion that, in adults, T-cell homeostasis is essentially formed from population movements mature T lymphocytes, and not, as previously believed, from the differentiation of hematopoietic stem cells differentiating into T lymphocytes via the thymus.
• mice have been described that a thymectomized mouse more than three days after birth lives normally with a normal number of T lymphocytes.
• a mouse thymectomized before D3 does not develop T lymphocytes, but if it is reinjected mature T lymphocytes, these develop and the mouse lives normally with a normal number of T lymphocytes.
• the principle of the invention is therefore particularly original because it proposes that the replacement of a patient's T lymphocytes does not necessarily go through a bone marrow or hematopoietic stem cell transplant (preceded by aplastic anemia (conditioning)) but can be carried out by a less invasive method, object of this invention, which is “the exchange of T lymphocytes”.
• the invention therefore offers a first advantage over previous techniques, which resides in the rapid reconstitution of immunity.
• the T lymphocytes injected into a patient allow a much faster expansion than the reconstitution from T lymphocytes derived from the differentiation of hematopoietic cells, which results in obtaining complete (or almost) replacement of the lymphocytes.
• T of the patient by a pool of T lymphocytes from those injected.
• the invention has another important advantage compared to prior techniques, which resides in its character of specificity.
• the present invention - has specificity with respect to immunopathological T lymphocytes, and - only modifies a portion of a subject's immune system.
• the invention also rests on the destruction of clones of immunopathological T lymphocytes, and therefore also has a character of stability insofar as the clones are deleted definitively.
• the present invention therefore offers numerous advantages over the previous methods, and has multiple applications.
• T lymphocytes 1) - a sample of autologous, syngeneic or allogenic T lymphocytes, the donor having T lymphocytes exhibiting immunity, antigenicity, age or other characteristics which it is desired to confer on the recipient,
• lymphocytes where appropriate the genetic transformation of the lymphocytes removed to give them a particular property, - where appropriate the modification (enrichment, depletion, depletion %) of this population of lymphocytes into a particular antigenic subclass, by depletion, enrichment or depletion in vitro or ex vivo, for example by immunoaffinity,
• the donor lymphocyte population if necessary genetically transformed or enriched by depletion of one or more unclassified subclasses can be administered to the recipient. extemporaneously, or frozen, for example by the technique described in patent application No. PCT / FR 97/00385.
• Freezing makes it possible to envisage a long-term treatment with a product which remains perfectly homogeneous over time. Similarly, the destruction of all or part of the patient's T lymphocytes is not necessarily required in all applications of the method.
• this type of treatment is numerous and cover all pathologies or physiological alterations involving the immune system.
• this type of treatment can be applied to autoimmune diseases, allergies, aging immune functions such as anergies, etc.
• This invention has at least two specificities:
• a first subject of the invention relates to the use of a population of T lymphocytes, for the preparation of a composition intended to be administered to a subject having undergone depletion of his T lymphocytes.
• the present invention is therefore based, on the one hand on the depletion of T lymphocytes of the recipient, on the other hand on the administration of a composition of T lymphocytes.
• a general diagram of this "exchange of T lymphocytes" is represented on the Figure 1.
• the T cell population used may include autologous, syngeneic, or allogenic T cells with respect to the subject.
• autologous refers to a population of cells from the subject to which they are administered. In this mode of implementation, the T cells are therefore removed from the subject before the depletion is carried out.
• the term “syngeneic” refers to a population of cells from a twin identical to the subject to which they are administered.
• allogenic designates a population of cells from another subject of the same species (related or not), that is to say from another human being in the case of the treatment of a human patient.
• lymphocytes can be obtained and prepared by any technique known to those skilled in the art.
• lymphocytes can be either isolated from blood, lymphoid organs or the like in a subject by conventional techniques (cytapheresis, density gradients, cell sorting, etc.) or obtained from stored banks.
• the lymphocytes are preferably obtained from mononuclear peripheral blood cells (PBMC) or from umbilical cord blood.
• PBMC mononuclear peripheral blood cells
• umbilical cord blood is an interesting source. Indeed, umbilical cord T lymphocytes have the advantage of understanding a very diverse repertoire, and of being able to support very many divisions.
• T lymphocytes can, in a particular embodiment of the invention, be taken from any allogenic donor, preferably chosen to have HLA compatibilities with the recipient, so that there can be cooperation between the antigen presenting cells of the recipient and the T lymphocytes of the donor.
• T lymphocytes can be taken from a donor chosen for a particular characteristic (for example, their proven capacity to respond to an infectious agent).
• the T lymphocyte population used include T lymphocytes representing a diversified repertoire. This makes it possible to promote a rapid reconstitution of the subject's immunity, and thus to avoid the persistence of significant immunosuppression in the subject.
• the peripheral blood contains about 2% of the total mature T cells of a subject. Furthermore, it is considered that for a given specific antigen, the frequency of clones of specific T lymphocytes is approximately one cell in 10 5 . Thus, from approximately 10 8 peripheral blood T lymphocytes, there is a population of T lymphocytes for which each antigenic specificity is represented by approximately 1000 cells. A population of this size therefore allows a completely satisfactory representation of the T lymphocyte repertoire. Preferably, during the sampling, it is desirable to obtain a quantity of T lymphocytes of the order of 10 6 to 10 9 , preferably 10 7 to about 10 8 cells, so as to cover a sufficient immunological repertoire.
• the population of T lymphocytes used for the administration comprises approximately 10 7 to 10 12 T cells, even more preferably between 10 8 and 10 11 , advantageously between 10 9 and 10 10 T cells. It is understood that these quantities can be adapted by a person skilled in the art.
• the population of T lymphocytes (Ly-T) used may include certain cells of different nature (not Ly-T).
• the population of T lymphocytes comprises at least 60% of Ly-T, preferably at least 80%, even more preferably at least 95% of Ly-T.
• the other cell types present can for example be B lymphocytes, or other blood cells, generally hematopoietic.
• the quality of the T lymphocyte population can be determined by any technique known to those skilled in the art, and in particular by labeling with different specific antibodies, and analysis by flow cytometry. Furthermore, the quality of the lymphocytes can also be verified by analysis of their repertoire by flow cytometry or by the immunoscope technique.
• the population of lymphocytes used is generally a population modified to improve the immune properties of the subject.
• the modification (s) made to the T cells can be of various natures.
• the population of T lymphocytes used comprises genetically modified T lymphocytes.
• lymphocytes comprise a nucleic acid not naturally present in lymphocytes in the unmodified state, or a nucleic acid present in an unnatural state in the lymphocytes (an artificially amplified sequence for example). In what follows, this nucleic acid will be designated heterologous nucleic acid.
• the genetically modified lymphocytes used in the context of the present invention comprise a heterologous nucleic acid comprising a suicide gene.
• suicide gene is understood to mean any nucleic acid coding for a toxic product, that is to say for a product (RNA, protein, etc.) capable of inducing destruction of the cell that contains it, by any mechanism.
• a product for example a protein
• conditional toxicity that is to say capable of transforming a normally inactive drug into a metabolite highly toxic to the cell.
• cellular toxicity is strictly controlled by the administration of the drug.
• the nucleic acid used is a nucleic acid whose action toxic specifically affects dividing cells.
• examples of this type of nucleic acid are in particular thymidine kinase (TK).
• HSV1-TK Herpes simplex virus type 1
• This enzyme unlike cellular thymidines kinases, is capable of phosphorylating different nucleoside analogs such as aciclovir (ACV) or ganciclovir (GCV) into monophosphate derivatives which will then be transformed into di- and triphosphates by cellular enzymes.
• ACCV aciclovir
• GCV ganciclovir
• These triphosphate compounds can then be incorporated by cellular polymerases into the DNA being elongated. This incorporation induces a termination of the elongation and triggers the apoptosis of these cells [11-18].
• the toxic doses of GCV necessary to destroy the cells expressing HSV1-TK are at least 100 times lower than those necessary to destroy the parental cells (approximately 10 times for ACV). Since GCV is much more effective than ACV in this system, it is the most widely used drug, although it requires administration by twice-daily infusion. The induction of cell death by blocking the extension of DNA implies that only dividing cells are affected by GCV, which has been confirmed experimentally. Thus, the HSV-1 TK gene has been used for gene therapy of cancer [5,6], and more recently for the destruction of ly-T causing pathological immune responses [1, 2,7-9] .
• This system therefore has three properties which are particularly advantageous for the present invention: (i) conditional toxicity dependent on the administration of a drug (a nucleoside analog) which will make it possible to control the destruction period of ly-T; (ii) toxicity restricted to dividing cells, property of activated ly-T; and (iii) great flexibility of use allowing different drug administration schemes.
• the prototype of the suicide gene is the HSV1-TK gene working with ganciclovir.
• Other thymidine kinases can also be used, such as thymidine kinase derived from Equine herpesvirus type IV. It can also be truncated TK genes such as the TK delta gene or an enzyme of human origin which has been mutated so as to acquire the properties of these viral enzymes.
• a nucleic acid encoding a conditional toxin constitutes a particularly preferred embodiment of the invention.
• the presence of this type of construct in the lymphocytes used for administration makes it possible to specifically control the immunopathological T responses.
• the presence of the suicide gene is used to destroy the T lymphocytes involved in the pathological immune responses and thus permanently remove the clones of lymphocytes responsible for these pathologies.
• the presence of the suicide gene is used to destroy the T lymphocytes engaged in a reaction of the graft against the host, and thus avoid the development of this type. pathologies.
• a patient suffering from a pathology mediated by an immunopathological T response is subjected to an efficient depletion of T lymphocytes (as described below), then is reconstituted with T lymphocytes.
• T lymphocytes can either be the T lymphocytes of the patient himself (autologous) after they have been transduced by a suicide gene (i.e. say genetically modified), or allogenic T lymphocytes, also modified.
• the mature T lymphocytes thus injected into the subject deprived of his own mature T lymphocytes will very quickly proliferate and expand in order to reconstitute the pool of mature T lymphocytes according to the homeostasis property of mature T lymphocytes to undergo an in vivo expansion of so that their number reaches the values before depletion.
• the rare Ly-T remaining in the subject will be able to multiply even less than the compartment dedicated to Ly-T will have been "filled” by the injected cells.
• the subject is treated with the drug capable of being transformed into toxic metabolite by the suicide gene used, which leads to a preferential elimination of only dividing T lymphocytes, that is to say only T cells activated and therefore responsible for the pathology.
• the invention also has the advantage of being able to be implemented even in the absence of knowledge, a priori, of the antigens involved in the development of the pathology concerned.
• the population of T lymphocytes used comprises immunologically modified T lymphocytes. More particularly, this immunological modification includes the modification in vitro (or ex vivo) of the T lymphocyte repertoire of said population. More preferably, this immunological modification includes the suppression in vitro or ex vivo of clones of lymphocytes involved in immunopathological responses and / or of subpopulations of T lymphocytes having particular immunological properties (CD4 + or CD8 +, TH1 or TH2. .).
• the population of T lymphocytes used therefore comprises a "hole" in the immunological repertoire.
• This mode of implementation is particularly suitable for situations in which the antigen (s) recognized by the immunopathological clones are known. Indeed, in this case, it is possible to carry out depletion in vitro or ex vivo on the population of T lymphocytes, by immunoaffinity using said antigens or fragments or homologs thereof for example. Such depletion can be carried out, for example, by passing the population of T lymphocytes in contact with a support on which said antigens or fragments or homologs are immobilized. Depletion of clones recognizing specific antigens involved in immunopathology can also be carried out after a transfer of suicide genes into T lymphocytes, if these are then stimulated in vitro in the presence of the antigen and of treatment with the conditional toxic (GCV). In the absence of a genetic modification, these clones of lymphocytes activated by the antigen in vitro can also be destroyed by antibodies coupled to toxin specifically recognizing molecules expressed by the T lymphocytes activated (receptors at 1 * 112).
• This mode of implementation can be carried out for example in the case of pathologies such as organ transplantation in which the alloantigens of the organ donor are involved.
• T lymphocytes can be implemented as well with an autologous or syngeneic population as with an allogenic population. In addition, it can also be combined with a genetic modification as described above.
• the population of T lymphocytes used comprises modified lymphocytes:
• the population of T lymphocytes used comprises allogenic T lymphocytes providing increased immunity.
• the T immune response is partly controlled by genetic factors of great diversity. Therefore, vis-à-vis a given antigen, some subjects are able to show appropriate (beneficial) responses while others are not. According to the invention, it is thus possible to replace the T lymphocytes of a subject sensitive to a given pathology by those of an insensitive subject.
• the T cells need not be immunologically modified.
• these T lymphocytes are genetically modified as described above.
• T lymphocytes are easily removed, isolable and cultivable cells, they can in fact be used simultaneously as a source to introduce (or reintroduce) proteins or absent or abnormal functions into a subject.
• T lymphocytes For the genetic modification of T lymphocytes, several approaches can be used, according to the techniques of a person skilled in the art.
• the lymphocytes are genetically modified using a viral vector.
• the heterologous nucleic acid is for example introduced into a viral vector, which is then used to infect a population of T lymphocytes as described above.
• viral vectors can be used, including retroviral or AAV vectors.
• the lymphocytes are genetically modified using a retroviral vector. Retroviruses are the vectors of choice for the transfer of genes into T lymphocytes because retroviral infection results in the stable integration of the genome into cells.
• retroviruses belonging to the family of oncoviruses, lentiviruses or spumaviruses.
• retroviruses belonging to the family of oncoviruses, lentiviruses or spumaviruses.
• oncoviruses mention may in particular be made of slow oncoviruses, not carrying an oncogene, such as for example MoMLV, ALV, BLV, or MMTV and rapid oncoviruses, such as RSV for example.
• oncogene such as for example MoMLV, ALV, BLV, or MMTV
• rapid oncoviruses such as RSV for example.
• lentivirus family mention may, for example, be HIV, SIV, FIV or CAEV.
• a recombinant retrovirus comprising an envelope of the GALV virus (retrovirus pseudotyped with GALV).
• GALV Gibbon Ape Leukemia Virus
• the recombinant virus used may contain expression regulatory sequences (promoters) specific for certain in T lymphocyte populations.
• promoters specific for certain in T lymphocyte populations.
• it may be desirable to express the nucleic acid only in certain populations of T cells such as, for example, CD4 + or CD8 + cells, or also cells called Th1 or Th2 for which specific markers have just been described in mice, allowing their separation.
• T cells such as, for example, CD4 + or CD8 + cells, or also cells called Th1 or Th2 for which specific markers have just been described in mice, allowing their separation.
• These cells being characterized by the type of cytokines they produce (for example IL2 or IFNg for Th1, and IL4 for Th2), it is possible to use the regulatory sequences of these different genes to control the expression of l heterologous nucleic acid (including the suicide gene).
• promoters controlling a specific expression in such or such population of T lymphocytes such as for example the promoter coding for the CD4 molecule.
• the lymphocytes are genetically modified by means of a retrovirus produced in an packaging line comprising a truncated pol gene.
• T lymphocytes capable of transducing T lymphocytes therefore allows an improvement in terms of safety.
• Infection of T lymphocytes by retroviral vectors can be carried out according to any technique known to a person skilled in the art (incubation with a retrovirus supernatant, co-culture T lymphocyte-cells retrovirus packaging, Transwell techniques, etc.).
• a particularly effective method has been described by Movassagh et al. (Cf Supra), comprising a possibly repeated centrifugation step. This method can advantageously be used for the genetic modification of T lymphocytes according to the invention.
• T lymphocyte population comprising at least 50%, preferably at least 65%, more preferably at least 80% of genetically modified T lymphocytes.
• a population of T lymphocytes is used, the rate of genetic modification of which is as close as possible to 100%.
• such levels can be obtained in vitro or ex vivo, using for example a GALV envelope, and if necessary under certain infection conditions (Movassagh et al., Supra).
• genetic modification since genetic modification is carried out in vitro, it is also possible to improve transduction levels by selecting the cells actually infected.
• various selection techniques known to those skilled in the art are available, and in particular the use of antibodies recognizing specific markers on the surface of infected cells, or the use of resistance genes (such as the resistance gene neomycin and the drug G418 for example), or compounds toxic to cells not expressing the transgene (ie, thymidine kinase).
• non-toxic molecule only for cells carrying the HSV1-TK gene.
• the method to find this molecule is to make a screen with a library of molecules known for their toxicity and to test those which are 1) modified by the enzyme HSV1-TK and 2) whose metabolites are no longer toxic.
• the advantage of such a method is that it does not need to transfer a second gene and that the selection only relates to unwanted cells.
• the selection is carried out using a marker gene, introduced into the retroviral vector, expressing a membrane protein. The presence of this protein thus allows selection by conventional separation techniques such as sorting by magnetic beads, columns, or sorting by flow cytometer.
• a gene like the one encoding the human Thy1 molecule is advantageous.
• a molecule such as the CD34 molecule which is absent from mature Ly-T because there are already cell selection systems carrying this surface molecule, these systems having already been validated for sorting cells and used in clinical.
• these markers can also include tag sequences, for example the c-myc tag.
• a membrane expression marker gene also has two additional advantages in the context of the present invention: (i) it makes it easy to follow genetically modified T lymphocytes when they have been injected, and above all (ii ) destroy them (even in the absence of division) thanks to the action of an antibody specifically directed against this molecule (if this proves necessary).
• the marker gene such as Thy-1
• so-called bicistronic vectors for which the toxic (eg, HSV1-TK) and marker (eg, Thy1) genes are separated by a IRES sequence.
• the population of T lymphocytes obtained, if necessary genetically modified and / or immunologically and / or having increased immunity, can be conditioned in any medium and any suitable device.
• the media which can be used are any culture medium for mammalian cells (RPMl, DMEM, etc.) or any other solution suitable for the conservation and / or storage of mammalian cells (saline solutions, buffers, etc.).
• the device used can be, for example, a tube, flask, box, ampoule, syringe, pocket, etc., preferably under sterile conditions suitable for pharmaceutical use.
• the cell composition can be used extemporaneously or stored (in the cold, frozen, or lyophilized, for example) for later use. By the way, as noted more far, banks of such cells can advantageously be produced, under the conditions of preparation and storage described above.
• the invention consists in an exchange of T lymphocytes from a subject, and therefore comprises the administration of mature T lymphocytes to a subject having undergone depletion of his own T lymphocytes.
• the subject Prior to administration, the subject is therefore subjected to a depletion step of his existing T lymphocytes.
• the population of T lymphocytes used is a population of autologous T lymphocytes, this population is prepared prior to depletion.
• Depletion can be done in different ways.
• conditioning including radiotherapy.
• depletion is more preferably carried out by treatment of the subject in the presence of one or more immunosuppressive agents, in particular in the presence of an immunosuppressive agent specific for T lymphocytes. Therefore, the subject is not subjected to aplasia total marrow and retains some immunity, despite T cell immunosuppression
• the immunosuppressive agent used is an "antilymphocytic serum", or one or more monoclonal antibodies specific for the surface molecules of T lymphocytes.
• an "antilymphocytic serum” or one or more monoclonal antibodies specific for the surface molecules of T lymphocytes.
• a serum or antibodies against CD3, CD4 and / or CD8 This type of immunosuppressants (serum or antibody depletants) has already been used in therapy.
• experiments carried out in mice and in humans show that it is possible to very effectively deploy an animal or a patient into mature T lymphocytes by these treatments (Muller et al., Transplantation 64 (1997) 1432; Caillat- Zucman et al., Transplantation 49 (1990) 156).
• T lymphocytes do not have to be complete for the exchange of T lymphocytes according to the invention to be effective. Thus, even if a certain percentage of T lymphocytes remains, it is unlikely that these will be enough to re-induce immunopathology. Generally, the immunosuppressive treatments described above lead to depletion greater than 90%, often 95%, which is entirely suitable for the present invention.
• composition of T lymphocytes can be administered in different ways and according to different protocols. It is desirable to administer T cells on a date such that treatments administered to patients for the purpose of destroying their own T cells have been eliminated, so that they cannot in turn attack the T cells which are to be injected. . Preferably, the composition is administered shortly after
• the treatments can be repeated if necessary, with the same cells, or even cells from different donors but according to the same therapeutic principle.
• Administration can advantageously be carried out by intravenous or intraarterial injection.
• the doses administered are generally between 10 8 and 10 11 T lymphocytes per subject. Furthermore, during the procedures described above for the preparation of T lymphocyte populations, it is possible to keep patient cells (normal and or transduced by the suicide gene) which can be used if necessary (stocks 1 and 2 in Figure 1). For example, if undesirable effects occur, it would still be possible to deplete the reinjected cells (for example allogenic) in order to then reinject the (normal) cells of the patient.
• the donor cells in the case of an allogenic exchange, can also be kept for later use if necessary (stocks 3 and 4 in FIG. 1). It is also possible that in a patient who has received transduced allogenic T lymphocytes whose GVH has been controlled, it is then possible to inject normal lymphocytes from the same donor without there being GVH (tolerance active). This would have the advantage of reinjecting cells which have not been cultured.
• the method of the invention also comprises a step of constituting banks of T cells, transduced or not, originating from the donor and from the recipient.
• the invention makes it possible to control the appearance or the development of immunopathologies, either by administration of the drug metabolized into toxic product (in the case of T lymphocytes genetically modified by a gene toxic), or simply by building up increased immunity in the subject.
• a product comprising:
• composition comprising a population of T lymphocytes as defined above, for a separate use or spaced over time, in particular for the control of immunopathological responses.
• the composition comprises a diversified repertoire of T lymphocytes.
• the composition comprises a population of T lymphocytes comprising a suicide gene
• the product of the invention also comprises a drug capable of being transformed into toxic metabolite by the suicide gene.
• the invention also relates to a process for the preparation of a composition of modified T lymphocytes comprising: - the removal of T lymphocytes from a subject, and
• the repertoire gap is created by depletion of T lymphocytes specific for antigens involved in pathologies.
• the invention also relates to any population of T lymphocytes taken from a subject and depleted of clones of lymphocytes specific for one or more antigens involved in one or more pathologies.
• the invention can also be implemented with any other type of compartment cell, that is to say any type of cell meeting the following criteria:
• Ly-T Ly-T or other circulating cells (satellite muscle cells).
• the applications of the invention are extremely numerous. They concern in particular, both for autologous and allogenic T lymphocytes modified according to the invention, all autoimmune and chronic inflammatory pathologies in the broad sense (rheumatoid arthritis, multiple sclerosis, etc.), viral infections (hepatitis A, B, C, HIV, etc.) or genetic diseases.
• the replacement of T lymphocytes also relates to organ or cell transplants (allogenic, even xenogenic) as well as all pathologies for which one may wish to re-inject modified lymphocytes to, for example, secrete a therapeutic protein.
• This system can also be advantageous in order to be able to control the immune response against a therapeutic protein administered for example to correct a genetic deficiency in this protein.
• T lymphocytes express a suicide gene, it is then possible to control this harmful immune response.
• autoimmune diseases called system diseases (ex: Systemic Lupus Erythematosus, Rheumatoid Arthritis, Polymyositis ”) are conditions with a clear immunological component as evidenced by the biological and histological investigations carried out in patients.
• system diseases ex: Systemic Lupus Erythematosus, Rheumatoid Arthritis, Polymyositis .
• the primum movens is not known and it seems that the origin of the pathology is multifactorial.
• the central element remains an inadequate immune response.
• this strategy leads to the deletion of clones of cells responsible for the pathology, and therefore in principle to lasting, even permanent, immunosuppression in the absence of central ly-T production.
• a patient suffering from rheumatoid arthritis receives, after depletion of his own T lymphocytes, an administration of a composition comprising his own T lymphocytes genetically modified to express a suicide gene (autologous composition) or allogenic T lymphocytes, modified or not. Then, during possible outbreaks of rheumatoid arthritis occurring after administration, treatment with a drug transformed into toxic metabolite by the suicide gene makes it possible to destroy the reactive clones and therefore to suppress the immunopathological response.
• a suicide gene autologous composition
• the present invention can also be used for the treatment of viral-induced immunopathologies.
• the immune response against infectious agents can often have immunopathological consequences, sometimes leading to death.
• the most classic example is that of the response to certain viruses responsible for hepatitis. These viruses replicate in hepatocytes and it is the destruction by the immune system of infected hepatocytes which leads to hepatitis, sometimes fatal.
• the host's immune response seems incapable of eradicating the virus, and on the contrary participates in the maintenance of chronic hepatitis. This is well illustrated by the evolution of so-called hepatitis C virus. While about two thirds of patients are able to get rid of the virus, the remaining third develops chronic hepatitis.
• the evolution of this chronic hepatitis is independent of the rate of viral replication and is, on the contrary, accompanied by biological signs testifying to a dysimmune response (for example frequent presence of anti-DNA antibodies or cryoglobulinemia).
• the present invention makes it possible to eliminate the clone (s) of active T lymphocytes responsible for immunopathology, and therefore to reduce the consequences of the infection on the host.
• the synthesis of IgE implicated in certain allergies is also under the control of T lymphocytes.
• the production of B lymphocytes is carried out from immature cells of the bone marrow and these cells have a much half-life. shorter than T lymphocytes. Therefore, it is also possible using the T lymphocyte exchange technique according to the invention to control the allergic responses, and more generally the antibody responses by controlling the T responses.
• the present invention can also be used for the treatment or prevention of Rejection of organ transplants.
• transplants of allogenic or xenogenic cells modified or not, so that they produce a factor for therapeutic purposes (eg cells of ⁇ islets of the pancreas, fibroblasts, etc.).
• this has been proposed in diseases as varied as diabetes, Parkinson's disease, and even in gene therapy in organoids.
• the main obstacle to such transplants remains the rejection of allô or xenogenic cells.
• numerous devices have been proposed in order to separate the grafted cells from the immune system. These systems range from microencapsulation to the insertion of cells into porous or semi-permeable materials, etc. Unfortunately, none of these systems has proven sufficiently effective to be offered in the clinic.
• the present invention now offers a new medical approach to promote the success of organ or cell transplants.
• an exchange of T lymphocytes is practiced in the recipient subject, which makes it possible, if necessary, to control any development of a possible immune response directed against the graft, by selective destruction of the alloreactive T lymphocytes.
• these therapies either by reimplanting in the recipient an organ or allogenic T cells and cells from the same donor, (and which are therefore tolerant to organ or transplanted cells), either to transplant an organism or cells from an allogeneic donor and to re-implant the patient's own genetically modified T lymphocytes in order to control the response against this organ or these cells.
• a particularly advantageous embodiment of the present invention lies in the treatment of immunopathologies in the context of organ or cell transplantation. More specifically, treatment refers to the prevention, total or partial, or reduction or elimination of immunopathologies mediated by T lymphocytes, which are responsible for transplant failures. Similarly, the invention can also be implemented for the treatment (prevention, reduction or total suppression, for example) of graft versus host disease (GVHD).
• GVHD graft versus host disease
• Bone marrow transplantation is a classic treatment in a large number of clinical situations, especially for many leukemias. The conventional treatment then relies on the conditioning of the donor, the aim of which is to eliminate the maximum number of tumor cells, and which has the consequence of creating a bone marrow aplasia requiring a bone marrow transplant.
• This bone marrow transplant is preferably performed with allogenic cells because these have proven that they can be responsible for a so-called “leukemia graft” effect which considerably limits the rate of relapses of leukemia. thus treated.
• allogenic T cells are also responsible for graft-against-host disease, which is still serious and sometimes fatal.
• the use of the present invention makes it possible to destroy the T cells responsible for GVH, and therefore to treat this immunopathology.
• T lymphocytes are a main target of infection by the HIV or certain genetic diseases.
• T lymphocytes are a main target of infection by the HIV or certain genetic diseases.
• HIV whose clinical course depends largely on the level of these lymphocytes in the infected patient.
• Some therapeutic strategies for HIV infection are based on the transfer of genetically engineered T cells to resist the virus.
• T lymphocytes are less susceptible to HIV infection due to spontaneous mutations in genes coding for proteins involved in the replicative cycle of the virus.
• the present invention makes it possible to take advantage of these genetic factors of great diversity, in particular by replacing the T lymphocytes of a subject sensitive to a pathology given by those of an insensitive subject.
• T lymphocytes can also be taken care of by an exchange of T lymphocytes according to the invention.
• the population of donor T cells can be used in the prevention or treatment of aging immune functions such as anergies.
• aging immune functions such as anergies.
• FIG. 1 Schematic representation of the principle of the Standard Exchange of T lymphocytes.
• Figure 2 Sensitivity to GCV of T cells expressing a thymidine kinase. The splenocytes were cultured for 2 days in the presence of ConA and increasing concentrations of GCV, then loaded overnight with tritiated TdR (a "uptake" of 100% is that observed in the absence of GCV). Triangles, squares and circles correspond to different forms of TK. The upper curve corresponds to the non-transduced T lymphocytes.
• FIG. 3 Protective effect of GVH of a 7-day treatment with GCV.
• FIG. 4 Pharmacogenetic control of lymphocytic choriomeningitis in mice expressing the suicide gene in populations of CD4 and CD8 lymphocytes.
• FVB or C57BL / 6 mice were irradiated and reconstituted by syngeneic bone marrow originating from FVB mice or from transgenic mice EPDTKL20, or by allogenic marrow.
• the recipients were infected by the intracerebral route with the lymphocytic choriomeningitis virus (10 4 pfu from LCMV strain Arm / 53b) and treated with GCV for 7 days (twice daily intraperitoneal injections of 100 mg / kg / j).
• Mice expressing the TK gene in their lymphocytes are protected, survive, have eliminated LCMV and have a high titer of anti-LCMV antibodies.
• transgenic mice expressing the HSV1-TK gene in ly-T. This expression is obtained using regulatory sequences derived from the gene encoding the CD4 molecule, which we had previously shown to lead to the specific expression of a transgene in lymphocytes
• transgenic mice expressing the HSV1-TK gene in T lymphocytes we have shown that it is perfectly possible to control the allogenic response responsible for the graft versus host reaction in these animals. We have thus demonstrated in principle this capacity to control the T lymphocytes responsible for GVH. It should also be mentioned that this therapeutic efficacy can be obtained by extremely short treatments with ganciclovir, and that we have shown that after these treatments not only pathology was avoided, but that the mouse had a response normal immune system.
• T HSV1-TK for the development of a model of graft versus host disease (GVH) after allogeneic bone marrow transplantation (GMO) [8].
• GVH graft versus host disease
• GMO bone marrow transplantation
• the purpose of this experiment is to demonstrate the efficiency of the exchange of genetically modified T lymphocytes on the control of a spontaneous autoimmune disease (MAI) represented here by the autoimmune diabetic mouse model NOD.
• MAI spontaneous autoimmune disease
• the exchange of T lymphocytes is carried out under conditions close to those envisaged in the clinic.
• the exchange of T lymphocytes is made with T lymphocytes allogenic to the recipient allowing the destruction of possible residual autoimmune cells.
• the exchange of T lymphocytes takes place with genetically modified T lymphocytes expressing the TK suicide gene.
• the control of GVH is based on the use of genetically modified T lymphocytes expressing the TK gene associated with treatment with ganciclovir (GCV). This system allows the destruction of TK T cells in division thus controlling the GVH.
• GCV ganciclovir
• T lymphocytes takes place after non-myelo-ablative conditioning of the NOD mice by irradiation of 8-9 Gy resulting in 100% survival on D 120 in such mice.
• the exchange of T lymphocytes is done by injection into the retro-orbital sinus of irradiated mice of 10 7 cells associated with a 7-day preventive treatment with ganciclovir, initiated at the same time as the transplant.
• a first control group consists of mice receiving 10 7 allogeneic bone marrow cells but no T lymphocytes. In these mice, the graft may be partially or completely rejected and the MAI may reappear.
• a second control group consists of mice receiving 10 7 bone marrow cells from NOD mice but not T lymphocytes.
• Table 1 Study of chimerism 180 days after the transplant.
• the hematopoietic reconstitution is evaluated after labeling the CSMNSPs with monoclonal Abs and analyzed by flow cytometry.
• the donor B and T cells of origin are determined by the expression of MHC class I molecules (H-2 q ).
• the recipient's B and T cells are determined by the expression of MHC class II molecules (JA k ).
• Table 2 Peri-insulitis and insulitis in NOD mice after exchange of T lymphocytes.
• the mouse pancreas is removed, fixed in Bouin's liquid and then placed in paraffin.
• the presence of lymphocyte infiltrates is evaluated on 4-micron sections after labeling with hematoxilin and eosin.
• mice remained normoglycemic, thus attesting to the absence of diabetes.
• recipient mice are reconstituted by allogenic T cells and no longer develop autoimmune disease. 1.3. Control of virus-induced immunopathology
• mice We wanted to apply the same system to the control of a reaction of immunopathology induced by a virus.
• the most classic experimental system is that of controlling the CML virus-induced encephalopathy in mice.
• mice We then used our mice to try to control the immune responses responsible for rejection of an organ graft.
• the model used is the one that can be considered the most physiological. It is a vascularized heart transplant in a heterotopic position in mice. When this allogeneic transplant is performed in untreated mice, rejection occurs regularly within a few days of the transplant. When the mice are treated with ganciclovir for a very short period (7 days from the day of the transplant) the heart is permanently tolerated and retains its vital functions as evidenced by its beating.
• the treatment begins with an ablation of the T lymphocytes.
• This ablation can be carried out for example by combining the administration of "antilymphocytic serum” (immunoglobulins capable of recognizing T lymphocytes and destroying them in the presence of the patient's complement which are usually produced rabbit for example by Mérieux laboratories).
• the amount of SAL that is administered and the duration of administration are dependent on the clinical data on the pharmaceutical batch and the manufacturer's recommendations.
• This treatment can be performed at the same time as the administration of other immunosuppressants such as cyclophosphamide and cyclosporine.
• the deletion of T lymphocytes can be analyzed in the peripheral blood by their standard count. Depletion of more than 90% of peripheral blood lymphocytes is preferable.
• T lymphocytes are in this case obtained from an allogenic donor exhibiting the maximum compatibility of the major histocompatibility antigens. This can for example be a first identical HLA for situations in which the family genetic risk of finding the same pathologies within the family is not too high.
• the reinjected cells have been previously transduced by a suicide gene such as the HSV1-TK gene and the reinjected cells contain at least 90% of transduced cells. In addition, all of the quality controls for these cells are carried out (phenotype, analysis of the repertoire).
• a gancyclovir treatment at the rate of 10 mg / kg twice a day is administered to the patient. If signs of GVHD occur earlier, treatment may be considered even earlier. The treatment lasts at least a week. At the end of this treatment, the patient remains followed, in particular to check the reconstitution of T lymphocytes.
• the signs indicative of a graft versus host reaction, such as skin damage, are also monitored. If such manifestations occur, curative treatment with gancyclovir is initiated.
• the treatment is repeated and terminated when the lymphocyte count has found values of at least 200 T lymphocytes per mm3.
• a GVH which could not be controlled by gancyclovir
• these T lymphocytes having for construction a membrane marker, an injection of the anti-marker antibody helps in the elimination of T lymphocytes, if necessary.
• the lymphocytes which are reinjected can be patient's lymphocytes similarly transduced by the HSV1-TK gene. Under these conditions, after reinjection of the cells, no treatment with gancyclovir is instituted. After having noted the reconstitution of the patient's T lymphocyte pool, when acute phases of disease outbreaks occur, gancyclovir treatment will be instituted as early as possible after identification of these outbreaks which may, for example, be arthritis outbreaks inflammatory for the treatment of rheumatoid arthritis or neurological and muscular damage in the case of multiple sclerosis.
• the lymphocytes can come from the organ donor. In this case they are tolerant to the graft and the procedure is therefore GCV treatment immediately after the administration of genetically modified T lymphocytes in order to suppress GVH.
• the immune system is subject to aging, which is characterized by poor regulation of the immune response such as the appearance of autoimmune diseases or the inability to mount an effective immune response against a new antigen.
• the treatment is preventive.
• T lymphocytes are taken from him. It is easy and safe to collect 10 8 lymphocytes during cytapheresis.
• This operation can be repeated quite often at intervals of the order of a few months.
• the T lymphocytes are cultured and amplified in order to reach 10 10 - 10 11 T lymphocytes.
• the cells are then distributed and frozen for several years.
• the T lymphocytes are thawed and reinjected into the subject.
• This reinjection can relate to all or part of the frozen T lymphocytes and can be preceded by a total or partial ablation of the circulating T lymphocytes.
• T lymphocytes it is entirely possible, for example to replace only one subclass of T lymphocytes with another subclass of T lymphocytes. For example, it is quite possible to selectively destroy only CD8 lymphocytes (or those carrying of a particular TCR) and to sort the T lymphocytes previously frozen in order to inject only the desired subclass of T lymphocytes, or even to selectively deploy the graft of such or such a subclass of T lymphocytes.
• T lymphocytes are then replaced by younger T lymphocytes which are better able to perform their functions.
• alloreactive T cells are destroyed and replaced by harmless T cells.
• a preventive therapy Indeed, it is really a "rejuvenation" of T lymphocytes and therefore of the immune system.

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• 1998
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